Project Summary/Abstract Rabies virus causes more than 50,000 deaths every year and is nearly 100% lethal if untreated. Rabies is endemic in natural reservoir populations of animals in the United States and throughout the world, and host- adapted rabies virus strains exist for many of these reservoir populations, including bats, skunks, and raccoons. The rabies virus glycoprotein (RabvG) is responsible for binding to receptors on host cells and is the major target of the antibody response and a major component of vaccines. Its structure, however, has not been determined and little is known about how host-adaptation mutations to RabvG affect receptor binding and infectivity. In order to design improved antiviral therapies and vaccines for rabies virus and to better understand rabies virus infection, it is necessary to determine the structure of RabvG and to thoroughly examine its binding interactions with receptors and antibodies. This project aims (1) to quantify the binding affinity between RabvG and its three cellular receptors; (2) to characterize antibody binding to RabvG and to determine what effects antibody binding has on RabvG/receptor complexes; and (3) to express and purify RabvG, alone or in complex with receptor or antibody, as a conformationally uniform population for structural determination via cryo- electron microscopy (cryo-EM) or tomography. We will conduct bio-layer interferometry experiments to quantify the binding affinity between RabvG and its three cellular receptors (the neural cell adhesion molecule (NCAM1), the p75 neurotrophin receptor (p75NTR), and the nicotinic acetylcholine receptor (nAChR)). We will compare the binding affinity of RabvG from different host-adapted rabies virus strains to receptors from corresponding and different host species, and then measure the infectivity of vesicular stomatitis virus (VSV) pseudotyped with RabvG on cells expressing the different host species receptors in order to determine if receptor binding affinity correlates with infectivity. We will also use bio-layer interferometry to determine relative antibody affinity and if antibodies can disrupt pre- formed RabvG/receptor complexes to neutralize virus. Finally, both of these receptor and antibody binding studies will inform engineering and optimization of RabvG for structural determination by cryo-EM. These experiments will yield new information about virus/receptor/antibody binding interactions that can be used to predict rabies virus spillover events and host range jumps as well as to improve immuno- prophylaxis treatments. Solving the RabvG structure will provide templates to better understand viral entry and antibody neutralization, yield a model for other lyssaviruses that threaten human health, and aid in further development of broadly protective vaccines.